Cancer is one of the three leading causes of death in industrialized nations. As treatment and preventative measures for infectious diseases and cardiovascular disease continue to improve, and the average life expectancy increases, cancer is likely to become the most common fatal disease. In developed countries, about one person in three receives a diagnosis of cancer during his or her lifetime and almost one in four dies from it.
Cancers are the progressive growth of the progeny of a single transformed cell. A tumor or neoplasm is a population of cells that exhibit uncontrolled proliferation without regard to normal bodily requirements. A malignant neoplasm or cancer is one that threatens life by invading and destroying adjacent tissue and/or by seeding (metastasizing) to distant sites. Malignant tumors are divided into carcinomas (which arise from epithelial precursor cells), sarcomas (which arise largely from mesenchymal tissues) and lymphomas (which arise from precursors of red and white blood cells). Therefore, curing cancer requires that all the malignant cells be removed or destroyed without killing the patient. Unfortunately, the overt manifestation and initial clinical presentation of cancer usually occur at a late stage in the disease process when the capacity for invasion has already been unleashed. By the time of diagnosis, a high proportion of patients have occult or even clinically detectable metastases. The capacity of conventional cytotoxic approaches to succeed in the face of this advanced, accelerating disease has, unfortunately, been limited (1,2). In contrast to the short time between disease presentation and established metastasis, the period of transition from hyperproliferative, but noninvasive disease (3-5) to invasive cancer may be 10 years or more in humans. For breast cancer, this period is estimated to average 6 years (3,4).
A major problem confronting cancer researchers in developing immunological weapons against this disease is simply that these cells closely resemble the normal lineages from which they arise. Thus, despite major advances in the understanding of the factors that lead to the development of cancer, progress in the clinical management of cancer remains limited. This is due in large part to the limited success of conventional therapy in the treatment of metastasis.
Early research revealed that mouse tumors displayed molecules that led to rejection of tumor cells when transplanted into syngeneic (i.e., genetically identical) animals. These molecules are “recognized” by T-cells in the recipient animal, and provoke a cytolytic T-cell response with lysis of the transplanted cells. This evidence was first obtained with tumors induced by chemical carcinogens. The antigens expressed by the tumors that elicited the T-cell response were found to be different for each tumor. This class of antigens has come to be known as “tumor specific transplantation antigens” or “TSTAs”. Following the observation of the presentation of such antigens when induced by chemical carcinogens, similar results were obtained when tumors were induced via ultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53:333-336 (1974).
A class of antigens has been recognized which are presented on the surface of tumor cells and are recognized by cytolytic T cells, leading to tumor cell lysis. This class of immunogenic antigens that arouse T-cell mediated immune reactions in the cancer-bearing host is known as “tumor rejection antigens” or “TRAs”. The extent to which these antigens have been studied, has been via cytolytic T cell characterization studies, in vitro i.e., the study of the identification of the antigen by a particular cytolytic T cell (“CTL” hereafter) subset. The subset proliferates upon recognition of the presented tumor rejection antigen, and the cells expressing the tumor rejection antigens are lysed. Characterization studies have identified CTL clones that specifically lyse cells expressing the tumor rejection antigens. Examples of this work may be found in Levy, et al., Adv. Cancer Res. 24:1-59 (1977); Boon, et al., J. Exp. Med. 152:1184-1193 (1980); Brunner, et al., J. Immunol. 124:1627-1634 (1980); Maryanski, et al., Eur. J. Immunol. 124:1627-1634 (1980); Maryanski, et al., Eur. J. Immunol. 12:406-412 (1982); Palladino, et al., Canc. Res. 47:5074-5079 (1987).
The immune system responds to cancer cells in complicated ways. There are two main types of immune cells that play a significant role in combating disease: B (or bone marrow-derived) lymphocytes (“B cells) produce antibodies to foreign antigens (which constitutes the part of the immune system known as humoral immunity); and T (or thymus-derived) lymphocytes (“T cells”) are involved in cell-mediated immunity. There are three main subclasses of T cells, namely, helper cells, cytotoxic cells and suppressor cells often referred to as CD4 Th cells, CD8 Tc cells and CD8 Ts cells, respectively, on account of their reactivity with a group (“cluster”) of monoclonal antibodies specific to a surface marker that identifies a particular lineage or differentiation stage. Thus, all leukocyte surface antigens whose structures are defined are given a “CD” (cluster of differentiation) designation, i.e., CD4 and CD8 respectively. The presence of a TRA on a tumor cell is recognized by the T cells and antigen processing cells as a “non-self” or foreign antigen. T cells react with foreign antigens via receptors on their surfaces. The human immune system contains millions of clones of T cells, each of which has distinctive surface receptors. The physical properties of these receptors confer specific binding capabilities and permit each of the several million clones of T cells in an individual to operate independently. The T cell receptor is capable of recognizing a particular antigen only when it is associated with a surface marker on an antigen-presenting cell (APC), such as a dendritic cell or a macrophage. The surface markers belong to a group of molecules known as the major histocompatibility complex (MHC). Explained in the context of cancer, a tumor rejection antigen is acquired and processed by APC. The APC processes the antigenic protein into shorter peptides called epitopes that generally range from about 8 to about 12 amino acids in length. If the peptides are presented on class I MHC proteins to CD8 T cells, then the epitopes are usually about 8 amino acids in length. If the peptides are presented on class II MHC molecules to CD4 T cells, then the epitopes are usually 9-12 amino acids in length. Binding of the T cell receptor to the epitope of the antigen on the antigen-presenting cell induces changes in the T cell that triggers a cell-mediated immune response.
Two signals are primarily responsible for inducing the T cell mediated response to an APC associated with an epitope of an antigen. A first signal results from the binding cross-linking of the T cell receptors with the epitope:MHC protein complex. A second, co-stimulatory signal is sent by “accessory” membrane molecules on the APC when bound by their receptors on the responding T cell. Subsequent to the resulting activation of T cells is the secretion of soluble intercellular messengers, known generically as “cytokines”, which regulate the amplitude or intensity and duration of the immune response. Cytokines include the group of biomolecules formerly known as lymphokines, monokines, interleukins and interferons (Essential Immunology, seventh edition, Blackwell Scientific Publications, Oxford, Great Britain, 1991, pp. 140-150). In this fashion, T cytotoxic cells that recognize and are specific to the tumor rejection antigen are stimulated and attack tumor cells that express the antigen.
Malignant tumors have been treated with chemotherapeutic agents that directly impair tumor cells or with immunotherapeutic agents that cause non-specific activation of immunity of a host. In recent years, researchers using tumors of animals, mainly mice, have revealed that tumors can be completely cured by enhancing an antigen-specific immune response to tumor-related antigens and/or tumor-specific antigens present in various tumor cells. The treatment has been conducted in the clinic by enhancing the antigen-specific immune response to these tumor-specific antigens. It is now known, however, that the immune response mediated by the T cells acts either protectively or in a suppressive manner depending upon whether T cytotoxic cells and T suppressor cells are activated. Thus, tumor cells can modulate anti-tumor immunity by expressing antigens that preferentially activate Ts cells or by secreting cytokines that directly suppress or induce secretion of suppressive cytokines by T-cells. That is, the activated CD8 T cells will either recognize and kill the tumor cell carrying the appropriate epitope on its MHC class I molecule, or it will recognize and become tolerant to the tumor cell, depending on the type of the stimulated CD8 cell, cytotoxic or suppressor, respectively.
Active immunization with some tumor antigens or irradiated, autologous tumor cells themselves has been shown in experimental animals to induce T lymphocyte-mediated immunity which protects the immunized mice from subsequent challenge with histocompatible tumor cells (6-8). In various preclinical studies (9), immunologic destruction of emerging tumors due to T lymphocyte recognition of tumor antigens has appeared to involve CD8+ cytotoxic T (Tc) cells, but CD4+ T helper 1 (Th1) cells have also been shown to be important (10). Within the last few years, a number of such antigens have been identified (8, 11) that appear to be encoded by genes with tumor-specific expression, expressed in normal cells, but which have developed point mutations in the tumor cell, 3) for differentiation antigens, or 4) which are over-expressed in certain tumors (12, 13). Many of these tumor antigenic markers will not serve as auto-immunogens when expressed in the host and, therefore, not elicit protective T lymphocyte responses (11,14). The differentiation antigens would normally not be expected to raise an immune response due to clonal deletion of auto reactive T lymphocytes. In some cases, they do because the site of normal expression of those genes is in immune-privileged tissues such as the testis or the eye (11).
The ideal tumor antigen for use in a vaccine or at which to direct immunotherapy would be one which is present on all tumor types, absent or masked in normal tissues, evolutionarily conserved, and its function required for the malignancy of the tumor cells. Such an immunogen would be less likely to be able to be down regulated or mutated and still have the tumor cells grow and metastasize optimally. Thus, if tumor cells used such mechanisms to evade the immune response to that immunogen (15), the tumor cells would be reducing their ability to thrive.
Applicants discovered that tumor cells express a common antigen which was originally called oncofetal antigen (OFA). This protein was detected in early to mid gestation fetal cells, hence the term “Oncofetal Antigen”. It is comprised of a single polypeptide chain of 295 amino acids and has a molecular weight of about 37-44 kDa. OFA was identified by Applicants to be a universal tumor specific transplantation antigen as it was detected on chemical or irradiation induced rodent tumors. All tumors that Applicants have tested were shown to express OFA (1, 43, 44). The tumors include chemically- and virally-induced sarcomas, X-irradiation-induced T cell lymphomas, and many tumors of inbred rodents reported by others to express only a unique, non-shared TSTA. Besides rodent tumors, approximately 500 human tumors representing most cancer types have been tested—all were found to express OFA (43-45). For example, OFA is also expressed by carcinomas of the breast, kidney, lung, colon, gastric mucosa, larynx, pharynx, ovary and prostate whereas normal tissues of the same types do not express OFA (43-45). OFA is believed to play an important role in tumor progression and has been implicated in tumor invasiveness, metastasis and growth.
Oncofetal antigen has recently been cloned. Complementary DNA sequence alignments have revealed 99% identity with another human protein called immature laminin receptor protein (iLRP). Hence, these two proteins are believed to be identical. (Hereinafter, the terms “OFA,” “iLRP,” “OFA/iLRP” and “iLRP/OFA” are used interchangeably.) The mature form of this laminin receptor appears to be a dimer of acylated immature 32 kDa laminin receptor protein (iLRP) (16). Although the mature 67 kDa form is on many normal cells as well as on tumor cells, there appears to be a preferential expression of the 32 kDa iLRP by fetal and tumor cells (17, 18). The iLRP is evolutionarily conserved (19). Indeed, the amino acid sequence of the human iLRP differs from that of murine iLRP by only four amino acids (20).
Tumor invasion of host tissues and trophoblastic penetration of the endometrium share common biological features. Both processes involve the invasion of basement membrane, an event that is initiated by adhesion of cancer or trophoblast cells to basement membrane components and particularly to laminin. Adhesion to laminin is mediated through a variety of cell surface receptors. Other investigators (Van den Brule F A, et al., Biochem. Biophys. Res. Commun. 201:388-393 (1994)), have shown that the 67 kD laminin receptor (67LR) and galectin-3 are inversely modulated as the invasive phenotype of cancer cells progresses, with up regulation of the former, and down regulation of the latter, respectively. These investigators found that the 67LR expression levels in the fetus increased from the 7th week of gestation to a maximum at the 12th week, when invasion is maximal, and then declined. Expression of galectin-3 was inversely modulated by the gestational age, with a minimum expression at the 12th week of gestation. A year earlier (1993), and 6 years before our identification of Oncofetal Antigen as iLRP, Applicants reported (in Coggin et al., Arch. Otolaryngol. Head Neck Surg. 119:1257-1266 (1993)) that based on the results of flow cytometry using different strains of mice, that the proportion of cells expressing OFA increased gradually during the gestational life of the fetus to reach its maximum levels (29% of the cells) at mid-gestation (day 13) and thereafter dropped gradually to 5% at day 18, whereas newborn mice did not show increased levels of expression of OFA.
The transition from in situ tumor growth to metastatic disease is defined by the ability of tumor cells of the primary site to invade local tissues and to cross tissue barriers. To initiate the metastatic process, cancer cells must adhere to extracellular matrix (ECM) components, secrete proteases which digest the dense matrix of type IV collagen, glycoproteins, and proteoglycans allowing them to invade the interstitial stroma and respond to factors inducing motility of the invasive cells (21). For distant metastases, intravasation requires tumor cell invasion of the subendothelial basement membrane of blood vessels using the same mechanisms (22). Several published experiments have suggested that tumor cell interaction with the laminin component of the ECM is important to the expression of the metastatic phenotype (23, 24). Upon binding of laminin by the immature form of the high affinity laminin receptor (iLRP), its expression and that of the laminin-binding α6β1 or 4 integrin are enhanced (25, 26). Thus, the stability of laminin binding by the tumor cells is enhanced. Besides this, the same step induces production and secretion of the collagenase IV matrix metalloproteinases (27, 28) required for digestion of the ECM to allow metastasis to occur. Increased expression of collagenase IV is seen in invasive colonic, gastric, ovarian, and thyroid adenocarcinomas while benign proliferative disorders of the breast and colon and normal colorectal and gastric mucosa have low or no staining for these proteases (29,30). Increased expression of iLRP is also seen in a wide variety of human adenocarcinomas, including those of the colon, breast, stomach, and liver (29, 31). Over-expression of iLRP is associated with poor prognosis in several types of tumor (32-35). In breast carcinoma, over-expression of iLRP correlates with early dissemination of the tumor cells to the bone marrow that further emphasizes the role of iLRP in the metastatic process (36). Experimental administration of anti-iLRP antibody or anti-laminin antibody at the time of tumor cell injection inhibits tumor metastasis (37-39).
OFA/iLRP is immunogenic. OFA/iLRP-specific T cells cloned from irradiated mice have been identified as Th1-type CD4+ T cells, which produce interferon-gamma, or cytotoxic T cells which secrete interferon-γ. Also, CD8+ suppressor T cells, which secrete IL-10 are induced. In addition, stimulating peripheral blood mononuclear cells from patients with breast cancer with autologous tumor cells resulted in the expansion of tumor reactive T cells. Analysis of these tumor reactive T-cells cloned by Applicants revealed that a substantial proportion of the clones showed reactivity against purified OFA/iLRP.
In more recent experiments, Applicants have observed that immunization of mice with syngeneic tumor cells expressing iLRP resulted in cross-reactive protective immunity against a spectrum of syngeneic tumors because they all express iLRP (6, 7). Immunization with iLRP:nitrocellulose particles produced distinct T and B cell mediated immunity depending on the dose of iLRP used. Thus, immunization with the intact iLRP protein can induce effector or regulatory T cells depending on the dose used.
The OFA/iLRP also activates Ts cells. They secrete IL-10. Ts cells prevent Tc cells from exhibiting cytotoxic activity against tumor cells. Once the concentration of iLRP reaches a certain optimal concentration, it induces IL-10-producing Ts cells that prevent Tc cells from killing antigen-positive target tumor cells. This phenomenon caused by an excess of the T cell immunogen, 37-44 kDaOFA, enables the immune system to suppress Tc-mediated immunity. In other words, it is an immuno-regulatory controlled measure that prevents over-production of Tc cells to any Tc-antigen. This immuno-regulation prevents anti-self Tc-mediated immunity and other anti-self immunity.
Rohrer et al. (40) showed that the apparent tumor-free, long-term survivors of fractionated, sublethal x-irradiation had developed iLRP-specific memory Th1 and Tc lymphocytes even though they showed no sign of lymphoma development. Approximately, half of the RFM mice that were irradiated died within 6 months after irradiation with metastatic thymic lymphoma (41). Besides the memory effector Th1 and Tc lymphocytes induced by iLRP during tumor development, non-cytotoxic, iLRP-specific, CD8+ T cells that secreted IL-10 upon antigen stimulation were also cloned from those long-term RFM mouse radiation survivors (40,42). The IL-10 inhibited Tc activity (42) and so these cells can dampen anti-tumor immunity of whatever specificity. We suggested that the time of appearance and/or the relative number of IL-10-secreting CD8 T lymphocytes compared to that of iLRP-specific Tc cells may have been a factor in determining whether an irradiated RFM mouse developed a thymic lymphoma and died from it subsequent to X-irradiation (43). In this regard, Applicants have observed that during breast or renal cell carcinoma development in humans, iLRP-specific Th1, Tc, and IL-10-secreting, CD8+ T (Ts) lymphocytes were clonable from the patients' peripheral blood (44, 45). Consistent with their view of the contribution of the Ts cells to tumor progression (43), Applicants have also found that breast cancer patients with the highest ratio of iLRP-specific Ts:Tc lymphocytes required a second surgery due to tumor recurrence (44). Thus, the frequency of the IL-10-secreting, iLRP-specific Ts lymphocytes in cancer patients may be used as a prognostic for clinical response to therapy (44). Such methods are a subject of U.S. Pat. No. 6,335,174.
Thus, while use of OFA/iLRP for cancer therapy and as a vaccine holds promise, it is tempered by the possibility that such uses will also trigger Ts-mediated immuno-regulation. In this regard, Rohrer et al., Mod. Asp. Immunobiol. 1(5):191-195 (2001), state that it is important to define the peptide epitopes which stimulate iLRP/OFA-specific Tc, Th and the IL-10 secreting Ts cells in order to determine if the epitopes which stimulate the Ts cells are different than and located on a different portion of the OFA protein than the epitopes that stimulate the Tc and/or Th cells.